Nanocellulose Hydrogel for Blood Typing Tests

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Nanocellulose Hydrogel for Blood Typing Tests Rodrigo Curvello, Llyza Mendoza, Heather McLiesh, Jim Manolios, Rico F. Tabor, and Gil Garnier ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00080 • Publication Date (Web): 17 May 2019 Downloaded from http://pubs.acs.org on May 19, 2019

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ACS Applied Bio Materials

Nanocellulose Hydrogel for Blood Typing Tests Rodrigo Curvello1, Llyza Mendoza1, Heather McLiesh1, Jim Manolios2, Rico F. Tabor3 and Gil Garnier1* 1Bioresource

Processing Research Institute of Australia (BioPRIA), Department of Chemical Engineering, Monash University, VIC 3800, Australia 2Haemokinesis 3School

Pty Ltd., Hallam, VIC 3803, Australia

of Chemistry, Monash University, VIC 3800, Australia

*E-mail: [email protected] Abstract The gel test is the most prevalent method for the forward and reverse blood typing tests. It relies on the controlled centrifugation of red blood cells (RBCs) and antibodies through a gel column. This noncontinuous matrix is currently based on microbeads that often lack sensitivity. For the first time, nanocellulose hydrogel is demonstrated as a sustainable and reliable medium for gel-based blood typing diagnostics. Gels with a minimum 0.3 wt.% TEMPO-oxidized cellulose nanofibres (0.92 mmol/g carboxyl content) separate agglutinated and individual RBCs in the forward test. The addition of glycine is able to balance the osmotic pressure and reduce haemolysis to 5%, whilst retaining the electrostatic repulsion responsible for the gel network structure and its rheological properties. For the reverse typing, cellulose nanofibers are chemically crosslinked with hexamethylenediamine (HMDA), increasing the gel yield point in 8-fold. Sodium chloride is added to achieve the osmolality found in the human plasma and limit cell lysis in 15%, without affect the gel colloidal stability. Nanocellulose hydrogel constitutes a performant, low cost and green soft material, providing clear and well-defined results for both blood grouping tests. Keywords: nanocellulose, hydrogel, blood typing, gel test, red blood cells

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Graphical Abstract

1. Introduction Red blood cells (RBCs) present different surface antigens on their external membrane. Depending on the antigens expressed, RBCs can be classified into a specific blood type in a considered blood group (BG) system1-3. This is named forward blood typing. Reverse typing, in contrast, identifies antibodies dissolved in plasma by observing their reaction with known (‘reagent’) red blood cells. Both tests are fundamental to determine the donor-recipient compatibility and avoid serious consequences due to immunological response from antigen-antibody interactions4-5. Blood typing is performed through a few robust techniques including the tube test, slide test, gel column and microplate 6. Recently, paperbased diagnostics have offered an efficient, economical and instantaneous alternative for blood typing 7-9.

The gel column, also referred to as gel card method, is a traditional technique established by Lapierre

et al in 1990 10. Thanks to its consistency, reproducibility and relative low cost, it is by far the most prevalent method used in blood banks. It is based on controlled centrifugation of red blood cells through a gel column made of continuous gel or beads 11. Briefly, cells and antisera are added at the top of a microtube and allowed to react. Positive antigen-antibody interaction forms RBC agglutinates that remain in the upper portion of the gel matrix after centrifugation. RBCs which have not agglutinated permeate through the gel to the bottom of the tube upon centrifugation, signalling a negative reaction (Figure 1a). A variety of antibodies are simultaneously tested in separate tubes within the same plastic card; the outcome is relatively stable for late analysis and does not require highly skilled operation. Also, small samples (50 µL 0.8% RBC suspension and 25 µL antisera or plasma) are used, and the reaction is relatively fast (10 minutes centrifugation) 10-11. The gels or beads used in the typing card are either made of dextran-acrylamide gels or glass beads dispersed in isotonic diluents or low ionic strength solutions 10, 12-13. Although efficient in most situations, these opaque and non-continuous materials may present low sensitivity in some tests, notably in the base of weak antibody grouping 14-15. Gels offering improved sensitivity, cell stability, and made from natural resources, while remaining low cost and ease of processing, would greatly improve a prevalent technique for blood analysis. However, the literature 2 ACS Paragon Plus Environment

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is scarce and there have not been reports of new materials applied to the gel test during the last 30 years. Here, we investigate nanocellulose hydrogels as a matrix for forward and reverse blood typing using the gel card method. Cellulose is a biodegradable, biocompatible, renewable, hydrophilic and easy to functionalize natural polymer; it is available on all the inhabitable continents7. TEMPO (2,2,6,6tetramethylpiperidine-1-oxyl)-oxidized cellulose forms nanofibers of narrow diameter distribution (2 to 5 nm), several microns in length, and functionalized at the C6 glycosidic carbon with hydrophilic carboxylate groups16. In water, the electrostatic repulsion of nanocellulose fibres gives origin to colloidal gels even at low concentrations (0.1 to 1 wt.%)

17-18.

These gels form an organized and

transparent mesh matrix structure which is suitable for separation of biomolecules and cells. The development of nanocellulose hydrogels for blood grouping is dictated by two requirements: first, gels must prevent cell lysis, and second, protect the integrity of the nanocellulose network structure to achieve reproducible and sensitive blood typing. Due to the highly hypotonic medium, the evaluation of additives is critical to increase RBC viability without affecting the gel colloidal stability. A series of sugars, neutral polymers and amino acids is therefore assessed to quantify their effects on gel rheology, red blood cell morphology and rates of haemolysis. The chemical crosslinking of TEMPOnanocellulose is also evaluated, in order to enhance the mechanical rigidity of the gel and prevent its collapse due to screening by charged molecules such as buffer salts.

2. Materials and methods 2.1. Materials Never dried bleached Eucalyptus Kraft (BEK) pulp of approximately 10 wt.% solids was supplied by Australian Paper, Maryvale, Australia. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), sodium bromide (NaBr), poly(ethylene) glycol (PEG, MW: 400) and N-(3-Dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDC) were purchased from Sigma-Aldrich. Hexamethylenediamine (HMDA) was purchased from Chem Supply. Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were diluted for solutions as required and were purchased from ACL Laboratories and Merck, respectively. 12 w/v% Sodium Hypochlorite (NaClO) and N-hydroxysulfosuccinimide (Sulfo-NHS) were purchased from Thermo Fisher Scientific. D-glucose, glycine, trehalose and sodium acetate (NaOAc) were all analytical grade and purchased from Merck and diluted as required. Sodium Chloride (NaCl) was bought from Sigma-Aldrich and diluted as required. AbtectcellTM III 3% and antisera –D (IgM) For Further Manufacture grade (FFMU) were purchased from Seqirus. Empty polypropylene gels card, AHG and neutral Stargel10 gels cards and centrifuge were kindly provided by Haemokinesis, Australia.



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2.2. TEMPO-mediated Oxidation TEMPO-mediated oxidation of BEK pulp produced nanocellulose gels

16.

A 4 wt.% BEK pulp

suspension containing proportional amounts of TEMPO and NaBr were prepared according to Saito, et al. 16. To initiate the oxidation, 12 w/v% NaClO (pH adjusted to 10) at 3.3 and 5 mmol NaClO/g fibre was added drop-wise. The reaction is maintained at pH 10 by adding 0.5M NaOH. The oxidation process was maintained until reaction termination –corresponding to a non-decreasing pH. Oxidised fibres were recovered and washed through filtration with a Buchner funnel and stored refrigerated (4°C). The oxidised pulp was then dispersed in deionised water to achieve a concentration of 0.1, 0.2 or 0.3 wt.%. The oxidised pulp suspension was then converted into gels via mechanical fibrillation through a high-pressure homogeniser (GEA Niro Soavi Homogeniser Panda) at 1000 bar.

2.3. Conductometric Titration The carboxylate group content was determined by conductometric titration as previously reported 16, 19.

Freeze-dried oxidised pulp samples (approx. 30 mg) were suspended in 40 mL deionised water.

Then, 40µL 1% NaCl was added to the suspended sample. The pH of the suspended sample was adjusted between 2.5 – 3 prior to titration with 0.01M NaOH using a Mettler Toledo T5 titrator. The conductivity of the sample was monitored throughout the titration progress. The carboxyl group content CC (mmol COO-Na+/g fibre) was determined as:

𝐶𝐶 =

𝑐(𝑉2 ― 𝑉1) 𝑤

× 1000

(1)

Where V1 and V2 are the amount of titrant (start and end of conductivity titration curve) required to neutralise the carboxylic groups (in L), c is the NaOH concentration (mol/L), and w is the sample weight (g).

2.4. Crosslinking of cellulose nanofibers TEMPO-oxidized nanocellulose fibres were crosslinked to HMDA via EDC/Sulfo-NHS coupling. Equal volumes of EDC/Sulfo-NHS/HMDA (300 mM) were added to the gel in a mole ratio 1:1 to the existing carboxylate group content. The gel was gently mixed by 5 minutes and then incubated at room temperature by 2 hours.

2.5. Additives for Increased Cell Survival A series of additives was blended with the nanocellulose gel to increase red blood cell survival. Solutions of sugars (1.5 M D-glucose and trehalose), amino acid (3M glycine) and salts (3M NaOAc 4 ACS Paragon Plus Environment

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and 0.9% NaCl) were added to the nanocellulose gel at two concentrations (0.15 and 0.45 mmol additive / g solution). Similarly, different volumes of PEG (5 and 10% v/v) were evaluated as an additive to the gel.

2.6. Rheology Testing The stability of nanocellulose gels with additives was tested by rheology. Rheological behaviour of the nanocellulose gels was tested using an Anton Paar MCR302 rheometer with a cone (0.997°) and plate geometry (49.975mm). All testing was performed at 25°C and a solvent trap was used to ensure temperature stability. Two types of rheological tests were performed: viscosity measurements and oscillatory strain sweep. Viscosity measurements were done between shear rates of 0.5 to 100s-1. Oscillatory strain sweep was performed between 0.01 to 100% strain at constant 1Hz frequency. All measurements were done in triplicates to ensure repeatability.

2.7. Crosslinking density Crosslinking density was calculated according to Flour-Rehner theory 20 as: 𝑛=

𝐺′ 𝑅𝑇

(2)

Where G’ (Pa) is the storage modulus, R is the gas constant (8.314 J.mol-1.K-1) and T is the temperature (298K).

2.8. Forward blood typing Microtubes were loaded with 50 µL of 0.1, 0.2 and 0.3 wt.% nanocellulose gel. The gel card was centrifuged at 75 G for 1 minute using a gel card centrifuge. After centrifugation, 1 µL of antiserum D (IgM) and 20 µL of 0.8% reagent red blood cells were placed on the top of the gel column. As the control, 1 µL of antiserum -D (IgM) and 50 µL of 0.8% reagent RBCs were placed in an AHG gel card. The gel card was centrifuged in single (unique centrifugation, without changes of speed) or bi-phase (two consecutive centrifugations, of the same interval each, with increasing of speed at the second stage) mode system for 5 minutes, at room temperature (Table 1). The gel card was photographed using a Sony Xperia Tablet kept in a light box (The Judge II, GretagMachbeth). Image processing was performed through intensity analysis method using ImageJ (National Institutes of Health), followed by data processing described in supplementary section (S17).



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Table 1. Acceleration and time of centrifugation for gels of different nanocellulose fibres concentrations. Nanofibres

Type

concentration (w/v)

Acceleration

Interval

(G)

(min)

0.1%

Single phase

90

5

0.2%

Single phase

370

5

0.3%

Bi-phase

825 and 1,125

5 (each phase)

2.9. Reverse blood typing Microtubes were loaded with 50 µL of 0.1, 0.2 and 0.3 wt.% nanocellulose gel or crosslinked gel. The gel card was centrifuged at 75 G for 1 minute using a gel card centrifuge (Haemokinesis). Cards loaded with crosslinked gel were incubated at room temperature 2 hours before use. Then, 10 µL of human plasma B and 20 µL of 0.8% reagent red blood cells were placed on the top of the gel column. As the control, 20 µL human plasma B and 50 µL of 0.8% reagent RBCs were placed in a neutral gel card. The gel card was incubated at room temperature for 5 minutes. The gel card was centrifuged in single (unique centrifugation, without changes of speed) or bi-phase (two consecutive centrifugations, for the same period each, with increasing of speed at the second stage) mode system for 5 minutes, at room temperature (Table 2). The gel card was photographed using a Sony Xperia Tablet kept in a light box (The Judge II, GretagMachbeth). Image processing was performed through intensity analysis method using ImageJ (National Institutes of Health), followed by data processing described in supplementary section (S17). Table 2. Acceleration and time of centrifugation for standard and crosslinked gels of different nanocellulose fibres concentrations. Nanofibres

Type

concentration (w/v)

Acceleration

Interval

(G)

(min)

0.1%

Single phase

90

5

0.2%

Single phase

370

5

0.3%

Bi-phase

825 and 1,125

5 (each phase)

0.3% crosslinked

Single phase

280

5

2.10.

Cell viability test

After the bi-phase centrifugation, 0.3% (w/v) nanocellulose gel and RBCs from negative results were harvested from each microtube and transferred to empty polypropylene tubes. Samples were resuspended in 1 mL of binding buffer 1X (BD Bioscience) for releasing RBCs and gently mixed. Red 6 ACS Paragon Plus Environment

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blood cells were labelled with 5 µL of Annexin V-FITC (BD Bioscience), according to the manufacturer instructions. Levels of haemolysis were assessed by analysis of particle size and exposure of phosphatidylserine via flow cytometry (Beckman Coulter Cytoflex), based on forward scatter channel (FSC) and fluorescein isothiocyanate (FITC) signal for 10,000 events. Data analysis was performed through CytExpert software (Beckman Coulter).

2.11.

Optical spectrometry

Samples containing 100 µL of ultrapure water, nanocellulose hydrogel and AHG/neutral beads were placed on a 96 well-plate and analysed by ultraviolet–visible spectroscopy (Infinite 200 PRO, Tecan). Samples were scanned for absorbance from 400 to 1000 nm. Data analysis was performed through Origin Pro 9.1 (OriginLab Corporation).

2.12.

Microscope analysis

A sample containing 2 µL of RBC suspension was harvested from nanocellulose gel and placed on the glass slide and covered with slip. Cell morphology was assessed by microscopy (Nikon Eclipse Ni-E Upright Microscope) and images were captured and analysed by NIS-Elements Advanced Research Imaging Software.

3. Results 3.1. Effects of fibre concentration and level of cellulose oxidation The feasibility of nanocellulose gels for forward blood typing was first evaluated and compared to a commercial AHG gel card. This is the most appropriate product for this test and contains anti-IgG. The effect of nanocellulose fibre content in gel on separating positive and negative reactions is shown in Figure 1b. Gel strength upon centrifugation is dictated by the fibre concentration as it influences the density of fibre entanglements, contact points and ion density which constitutes the network. Gels containing 0.1–0.2 wt.% nanocellulose do not possess the required network fibre density to distinguish a positive from a negative reaction. Agglutinated and non-agglutinated cells migrate through the 0.1 wt.% gel and do not clearly segregate positive and negative results. The same effect is observed for the 0.2 wt.% gel, besides extensive haemolysis (cell rupture) occurring for the negative sample. A minimum of 0.3 wt.% provides the desired visual differences between positive and negative reactions. Due to the column transparency, the exact region of agglutination can be easily identified. However, cell lysis is still detectable. A carboxylate concentration higher than 0.92 mmol/g was found essential to provide the desired electrostatic repulsion between fibres (Figure S2). Microscopy analysis shows that positive reactions triggered red blood cell agglutination (Figure 1c), resulting in cells acquiring a polygonal 7 ACS Paragon Plus Environment


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shape in closed packed configuration, while individual cells from negative reactions are seen in their native pseudo-spherical morphology (Figure 1d). A sample of 0.3 wt.% nanocellulose hydrogel is illustrated in Figure 1e. Optical spectrometry demonstrates the transparency of the cellulose gel, whilst opacity of AHG beads is characterized by maximum absorption at 600-700 nm (Figure S3).

Figure 1. (a) The gel method for forward blood typing relies on the controlled centrifugation of RBCs and antibodies through a gel column. Positive antigen-antibody interactions promote haemagglutination, where agglutinates become trapped in the upper region of the gel matrix after centrifugation. Conversely, individual RBCs diffuse to the bottom of the tube upon centrifugation, indicating a negative result. (b) 0.1, 0.2 and 0.3 wt.% nanocellulose gels were tested to determine the ideal solids content for reliable blood typing. Gels at 0.1 and 0.2 wt. % did not provide the required fibre network density to distinguish a positive from a negative reaction. 0.3 wt.% cellulose fibres offered the desired separation. However, significant haemolysis was identified. Results represent typical experiments performed in triplicate (n=3) - refer to Figures S1 and S4. (c) and (d) Optical microscope analysis of agglutinated and individual red blood cells harvested in 0.3 wt.% nanocellulose gel demonstrated the occurrence of antigen-antibody reaction for positives and its absence in negatives. (e) Illustrative sample of 0.3 wt.% nanocellulose hydrogel in a petri dish.

3.2. Effects of additives on nanocellulose gels A series of model salts, sugars, amino acids and neutral polymer additives was tested with the aim to decrease red blood cell lysis without affecting the nanocellulose gel structure. The state of RBC agglutination is assessed by quantitative image analysis for each microtube. Most of the additives tested produce a clear and well-defined result for the positive reactions in forward blood typing, trapping the agglutination in the upper region of the gel column. For positive tests, peaks of red intensity are detected 8 ACS Paragon Plus Environment

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at distances of 1.0 to 1.1 cm from the bottom of the microtube. Such results resemble those obtained with the current commercial gel card, used as a reference (Figure 1b). In contrast, non-agglutinated red blood cells formed a distinct pellet only in a few gel formulations, corresponding to high redness intensity peaks at 0 to 0.1 cm from the bottom of the column. The gel containing 0.45 mmol/g D-glucose (Figure 2c-d) and trehalose (Figure 2e-f) exhibit a reddish column for the negative reactions and prevent sedimentation of RBCs. This behaviour is also identified in the 0.15 mmol/g trehalose gel (Figure S6e-f). For diagnostics, the dispersion of red cells through the gel could be interpreted as false positive or a mixed result. Similarly, non-agglutinated cells centrifuged in 10% (v/v) PEG-nanocellulose gel also disperse through the material and do not pellet at the bottom (Figure 2g-h). A few cells are at the bottom of the lower concentration of the gel column where glycine is used as the osmotic additive (Figure S6i-j), and weaker agglutination of RBCs is noticed in the 0.15 mmol/g D-glucose-gel (Figure S6c-d). Clean and well-defined flat red lines from positive reactions, as well as RBCs fully sedimented at the bottom for the negative test are seen for gels containing 0.45 mmol/g glycine (Figure 2i-j). For both of the inorganic salts tested (NaCl and NaOAc), gel phase separation with excluded water is observed, similarly to previous reports 18. For this reason, the loading of salt-containing gels in the microtubes was not possible, preventing full testing of both.



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Figure 2. RBC agglutination for positive and negative tests in 0.3 wt.% nanocellulose gel containing (a-b) non-additives, (cd) D-glucose, (e-f) trehalose, (g-h) PEG, and (i-j) glycine at 0.45 mmol/g, and 10% (v/v) for PEG. Agglutinated RBCs are trapped in the upper region of the gel column. Individual cells from negative reactions form a pellet at the bottom. Some gel formulations prevent RBC sedimentation. Graph curves indicate the location of red blood cells in the different regions of the gel column, based on analysis of red intensity. Results represent typical experiment performed in triplicate (n= 3) - refer to Figures S5 and S7.

3.3. Effect of additives on gel rheology Rheology was employed in order to understand the influence of salts, sugars, proteins, and polymers on gel network stability and mechanics. The viscosity–shear rate relationship is shown for gels of 0.1 and 0.3 wt.% nanocellulose content in Figure 3a. Pure nanocellulose gels possess a two-phase, shearthinning behaviour. The addition of trehalose, D-glucose and glycine at all concentrations tested had a minimal effect on viscosity, apparently mostly due to dilution. Indeed, gels retain their distinct rheological behaviour, as exemplified by glycine gels in Figure 3b. Both D-glucose and trehalosecontaining cellulose gels present very similar results (Figure S8a-b). The gel viscosity is only affected by adding polyethylene glycol (PEG), a neutral low molecular weight polymer. At high PEG concentrations (10% v/v), viscosity at low shear rates is slightly higher than for the pure gel (Figure S8c). Dynamic strain measurements show that the additives do not significantly impact on the nanocellulose viscoelasticity. Nanocellulose network strength, characterised by the elastic modulus (G’), is also influenced by dilution. As illustrated in Figure 3c, adding increasing amounts of trehalose, D-glucose, and glycine lowers G’. However, the addition of PEG, particularly at 10% (v/v), results in greater storage of elastic energy within the network, demonstrated by the increase in G’. This indicates stronger reticulation and a higher netpoints density (50.25 mmol/m3). For the other additives, the 10 ACS Paragon Plus Environment

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crosslinking density of the gel is not affected and is maintained between 20-30 mmol/m3 as show in Table 3. Loss and storage modulus curves show that glycine (Figure 3e-f) as well as the other additives (Figure S9) do not affect the viscoelastic curve of nanocellulose gels (Figure 3d). Tan δ, defined as the ratio of loss to storage moduli (G”/G’), shows that the modified nanocellulose gels maintained their elastic dominated characteristics over the same range (Figure S10). Cross-over points, wherein the gel yields and displays a viscous dominated behaviour, vary slightly for the different additives. The pure and diluted nanocellulose gels yield at lower strains than the modified ones. The addition of a high volume of PEG results in gels yielding at the highest shear. Addition of NaCl and NaOAc promoted gel collapse, and consequently, rheology for salt-containing gels is not measurable.

Figure 3. Viscosity curves of nanocellulose gels as a function of nanocellulose concentration. Viscosity–shear rate curves of nanocellulose gels at: (a) different fibre concentration, and upon the addition of (b) glycine at 0.15mmol and 0.45mmol/g against an unmodified gel and a gel diluted with water. (c) Effect of additives on the nanocellulose gels storage modulus (G’) measured from the linear viscoelastic region (LVR) of dynamic strain measurements (25°C, 1Hz). Storage (G’) and loss (G”) modulus versus strain (25°C, 1 Hz) for: (d) 0.3 wt.% nanocellulose gel, and (e) 0.15 and (f) 0.45 mmol/g glycine-containing gels. Measurements were performed at 25°C (n=3).

Table 3. Crosslinking density for 0.3 wt.% nanocellulose hydrogel and additives Hydrogel 0.3 wt.% Diluted 0.3 wt.% Glu 0.15 Glu 0.45

Crosslinking Density (mmol/m3)

41.98 ± 6.22 22.14 ± 1.24 30.78 ± 2.58 21.84 ± 2.24 11

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Tre 0.15 Tre 0.45 PEG 5% PEG 10% Gly 0.15 Gly 0.45

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29.09 ± 1.34 23.92 ± 1.99 34.46 ± 2.25 50.25 ± 3.03 33.69 ± 1.89 26.53 ± 2.14

3.4. RBC morphological changes Independently of the gel additive, morphological analysis shows positive reactions produce strong red blood cell agglutination in the forward typing tests. The cells are closely packed and for some, the RBC edges become indistinguishable. In contrast, the individual RBCs (from negative reactions) exhibit differences in morphology and size according to the gel additive (Figure 4a). D-glucose and trehalose at lower concentration induce RBCs to a spherical shape of average diameter 5.8 and 6.2 µm, respectively. At 0.45 mmol/g, the monosaccharide (D-glucose) induces RBCs to adopt the discoid-like morphology with a diameter of 6.7 µm. The disaccharide (trehalose) at higher concentration promotes cell transition to the spherical shape, with average diameter decreasing to 5 µm. For the gel column with 5% (v/v) PEG, RBCs acquire the biconcave morphology with diameter 8.4 µm; cells agglomeration is also observed. At higher PEG concentrations, the red blood cells shrink and size measurement is not possible. Similarly, with 0.45 mmol/g glycine, the gel induces red blood cells to adopt the biconcave shape; a few echinocytic RBCs are also produced. RBCs become spherical at lower glycine concentrations with cell size of 5.3 µm (Figure 4b).

3.5. Red blood cell viability Cell viability was determined based on occurrence of lysis by irreversible cell membrane injury. This is indicated by the externalization of phosphatidylserine (PS) on the red blood cell membrane derived from its damage when contacting the gel. Approximately 28% of RBCs undergo haemolysis once in contact with the standard nanocellulose gel (Figure 4c). A higher number of lysed cells, comprising 16% of the total population, is only observed in the D-glucose–nanocellulose gel (0.15 mmol/g). The second highest rate of haemolysis is identified in 10% (v/v) PEG-containing gel, where 10% of RBCs are stained by Annexin-V (antibody against PS). For all of the other additives, a maximum of 5% of RBCs have their membrane permanently damaged. Glycine massively reduced haemolysis, without damaging the cell membrane, keeping its levels similar to the control (3%).


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3.6. Effects of additives on gel osmolality Osmolality is defined as the concentration of osmotically active substances (osmolytes) per kilo of solvent. The RBCs survival is directly dependent on this parameter, once hypotonic media easily triggers haemolysis. In contrast, hypertonic medium modifies the red cell morphology with possible damages to the membrane, preventing its sedimentation. Therefore, the osmolality was measured for the different gel formulations (Figure 4d). For the standard gel, the concentration of osmoles is zero, as no osmolyte was present. Adding the stabilizing molecules studied increase osmolality from 130 to 713 mOsmol/kg. Glycine increased gel osmolality as high as 354 mOsmol/kg. PEG and D-glucose have the same effect on the gel at higher concentrations (5% (v/v) and 0.45 mmol/g). However, trehalose had a much lower effect on osmolality than the other additives. The highest osmolality of 713 mOsmol/kg was achieved for 10% (v/v) PEG–nanocellulose gel.

Figure 4. (a) Optical microscope analysis of agglutinated and individual red blood cells harvested in nanocellulose gel containing D-glucose, trehalose and glycine at 0.15 and 0.45 mmol/g, and PEG at 5 and 10% (v/v). Results represent experiments performed in triplicate (n= 3). (b) Diameter of individual red blood cells harvested from nanocellulose gels containing D-glucose, trehalose and glycine at 0.15 and 0.45 mmol/g, and PEG at 5 and 10% (v/v). Results represent the mean of experiments performed in sextuplate (n= 6). (c) Flow cytometry analysis of lysed RBC harvested in 0.3 wt.% nanocellulose gel combined to different additives. (d) Osmolality of nanocellulose gels containing D-glucose, trehalose and glycine at 0.15 and 0.45 mmol/g, and PEG at 5 and 10% (v/v). Results represent the mean of experiments performed in triplicate (n= 3, SD = ± 4 mOsmol/kg).

3.7. Crosslinking cellulose nanofibers for reverse typing Similarly to the forward test, the effect of nanocellulose fibre content in the gel was evaluated for reverse typing. Reverse typing is used to type antibody content in blood plasma; it provides the 13 ACS Paragon Plus Environment


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complementary analysis of red blood cell antigen typing. A commercial neutral gel card was used as control. It is the most suitable product for the reverse test. For 0.1 and 0.2 wt.% nanocellulose gels, both positive and negative reactions formed pellets at the bottom of the column after centrifugation. Gels containing at least 0.3 wt.% nanocellulose provided clear differences between positive and negative results. However, besides the occurrence of haemolysis, the 0.3 wt% gel lacked reproducibility (Figure 5a). Although the addition of glycine balances the gel tonicity and prevents cell lysis, the instance of false positives or negatives still occurs. The cellulose nanofibers were then chemically crosslinked with hexamethylenediamine (HMDA) to enhance the network stability of the gel. The 0.3 wt.% crosslinked nanocellulose gels offered distinguishable positives and negatives for reverse blood typing (Figure 5b). Red cell agglutination was located at 0.6–0.8 cm height, while individual cells underwent lysis, which resulted in a reddish gel column. Once crosslinked, the gel tonicity can be easily balanced with sodium chloride (NaCl) at a concentration of 0.9 wt%, without affecting the colloidal stability of the gel. For the isotonic crosslinked gel, agglutinated RBCs were trapped at 0.9 cm height. In contrast, nonagglutinated cells are readily centrifuged to form a suitable pellet at the bottom of the gel column. The forward typing for this blood sample was performed, matching with the result obtained on the back grouping (Figure S15). Microscope analysis showed that RBCs were well agglutinated in the case of positive antibody–antigen reaction. Individual biconcave and spherical red cells were seen in the negative result (Figure 5c). The gel was measured to have an osmolality of 0 and 135 mOsmol/kg in the absence and the presence of NaCl, respectively. After centrifugation, the gel was mixed with the human plasma and PBS, which elevated the osmolality to 248 mOsmol/kg (Figure 5d). For this reason, the rate of haemolysis decreased to 15% in the crosslinked gel containing salts, in contrast to 30% and 22% cell lysis in the 0.3 wt.% standard and crosslinked gel, respectively (Figure 5e). Rheological analysis showed that crosslinked nanocellulose gel maintained its original the shear-thinning behaviour (Figure 5f). Due to the covalent bonds between the fibres, the gels exhibit increased viscoelasticity, in the form of higher values of G’ and G”, when compared to non-crosslinked samples (Figure S16), with yield point 8 times higher (Figure 5g). The crosslinking density increased to 436 and 432 mmol/m3 for the hydrogel in presence and absence of NaCl, respectively.


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Figure 5. (a) Nanocellulose gels were tested to determine the optimal solids content for reliable reverse blood typing. Gels at 0.1 and 0.2 wt. % did not provide the required fibre network density to distinguish a positive from a negative reaction. A minimum of 0.3 wt.% cellulose fibres offered the desired condition; however, instances of false positives and negatives were noticed. Graphs indicate the intensity analysis of red blood cells in the gel column. (b) Cellulose nanofibers in 0.3 wt.% gels were chemically crosslinked with HDMA, enhancing reproducibility. NaCl 0.9% was added to the gel to balance tonicity. Results represent typical experiments performed in triplicate (n= 3) - refer to Figures S11-14. (c) Optical Microscope analysis of agglutinated and individual red blood cells harvested in 0.3 wt.% standard and crosslinked nanocellulose gel demonstrated the occurrence of antigen-antibody reaction for positives and its absence in negatives. (d) Flow cytometry analysis of lysed RBC harvested in 0.3 wt.% standard and crosslinked nanocellulose gel, in absence and combined to NaCl 0.9%. (e) Osmolality of 0.3 wt.% standard and crosslinked nanocellulose gels containing NaCl. Results represent the mean of experiments performed



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in triplicate (n= 3, SD = ± 4 mOsmol/kg). (f) Viscosity-Shear rate curves of 0.3 wt.% standard and crosslinked nanocellulose gels (n =3). (g) Stress–shear strain curve for non-crosslinked and crosslinked nanocellulose gels (n =3).

4. Discussion A reliable forward and reverse blood typing test need a clear visualization of RBCs, well differentiating agglutination from non-agglutination and preventing cell lysis. Commercial gel cards are currently made of opaque or colourful beads suspended in a buffer. Nanocellulose hydrogel, in contrast, forms a transparent continuous column. The trapped haemagglutination and individual cells can be easily identified on the top or bottom of the gel, respectively. Therefore, lower volumes of blood are required in the cellulose-based test which still reveals clear and very well-defined positive and negative results. Second, beads-based gels are not static and these microparticles migrate within the gel card well during testing, even after centrifugation. Disturbing its dynamic organisation requires waiting for sedimentation to occur, prior to performing typing. In addition, weak and small agglutinates achieved in the test can be lost in the heterogeneous material mixture formed by cells and beads. Thus, retaining colloidal stability of the nanocellulose gel is also required to ensure a homogeneous matrix that can provide constant and extremely reproducible separation. As nanocellulose gel is mainly composed of water (99.7%), erythrocytes burst upon contact with the hydrogel driven by a massive difference in tonicity. For this reason, red cells must not lyse within the cellulose matrix during centrifugation. This can be overcome by the inclusion of additives that prevent haemolysis while also preserving the gel stability through electrostatic interactions. If required, covalent bonding between the nanofibers can first be performed. Sugars, amino acids and neutral polymers can increase nanocellulose gel osmolality without collapsing the gel structure of nanofibers

21-22.

However, due to the different mechanisms of

interaction between RBCs and additives 23, haemolysis rates and cell morphology are diverse. D-glucose and trehalose do not dissociate in solution; they do not influence the balance of physical entanglement of nanofibres. The monosaccharide is not effective in regulating the cell lysis, allowing haemolysis of 16% of cells at lower concentration (0.15 mmol/g). According to literature, Dglucose oxidises as soon as absorbed by RBCs and can promote membrane peroxidation

24-25.

This

renders the spherical cell osmotically fragile and susceptible to injuries from the mechanical force of centrifugation. The trehalose-containing gel, in contrast, presents similar osmolality as D-glucose, triggers the same cell shape and exhibits much lower lysis with rates as low as 4%. However, this disaccharide is not permeable to the human RBC membrane and accumulates around the cells. It promotes RBC agglomeration, leading to false positive results. Thus, neither sugar is appropriate as an additive for the gel in a blood typing test. PEG has been traditionally used in low ionic strength saline (LISS) solutions to enhance weak antigen-antibody reaction, but its application is controversial and concentration-dependent 26. The complete coating of RBCs with long PEG chains would mask their antigens, preventing interactions with larger biomolecules such as antibodies 27-28. This could reflect in 16 ACS Paragon Plus Environment

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false negative results and make the test unreliable. Moreover, PEG at higher concentration (10% v/v) strongly increases osmolality up to 712 mOsmol/kg. This very hypertonic gel shrinks the RBCs and prevents their proper sedimentation. For this reason, even though 5% (v/v) PEG induces the expected RBC morphology (biconcave) and reduces haemolysis, this additive is not suitable for blood typing. Glycine presents the best results by increasing the osmolality to levels similar to those found in the human plasma (260–300 mOsmol/kg) 29. At 0.45 mmol/g glycine, RBCs acquired a stable biconcave or echinocytic morphology and haemolysis decreased to levels as low as 4%. A similar incidence of lysis is obtained even at the lower concentration. Since it is a natural osmolyte, glycine is easily released or uptaken by the cell according to the medium’s tonicity 30. This mechanism prevents sudden variation of the cell volume, especially when in presence of hypotonic solutions

31.

For this reason, glycine is

commonly found in LISS solutions, acting as an osmoprotector of cells without affecting the ionic strength 32. It makes this amino acid fully compatible with TEMPO-oxidized nanofibers, not affecting the electric double layer responsible for maintaining the gel network structure. Other amino acids, mainly those with charged and polar side chains, would affect the colloidal stability and trigger gel collapse. The same effect would be expected from salts, such as NaCl and NaOAc, due to their dissociation and further interaction of their ions with the cellulose nanofibres 18, 33-34. Similarly to ions derived from salts, the electrical charges of the proteins and peptides present in plasma also affect the electrostatic gel properties. In order to overcome this restriction in the reverse blood typing test, cellulose nanofibers were covalently crosslinked with HDMA by EDC/Sulfo-NHS coupling. The diamine behaves as a link between the existing carboxylate groups in TEMPO-oxidized cellulose nanofibres. Due to the amide bonds formed, the network structure of nanofibres becomes highly stable and enhances the gel reproducibility. Once crosslinked, the cellulose hydrogel is suitable for reverse blood typing, providing clear and distinct positive and negative results. In this scenario, glycine is not a suitable osmolyte, since its carboxyl and amine groups could react. The balance of tonicity can then rely on saline solutions, such as NaCl 0.9 wt.%. Indeed, salt-containing crosslinked gels increase the gel osmolality to desired values and minimize haemolysis rates as expected. Higher concentrations of salts would reduce lysis to even lower levels. Nonetheless, the effects on the red cell morphology might demand different conditions of centrifugation (time and acceleration).

5. Conclusion We report nanocellulose hydrogels as a novel and performant matrix for forward and reverse blood typing tests based on the predominant gel card method. There is a need for renewable, biodegradable and low-cost materials to engineer a new generation of high-performance biodiagnostics with improved sensitivity and reproducibility, allied to sustainability. Indeed, this field has not received any update for the last 30 years. Here, we developed nanocellulose gel as a versatile matrix to visualize the antibody-triggered agglutination and non-agglutination of red-blood cells to communicate positive 17 ACS Paragon Plus Environment


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and negative blood typing tests, respectively. A reliable separation is obtained in hydrogels containing 0.3 wt.% nanocellulose (99.7% water). Maintaining the red blood cells in an isotonic environment is essential to prevent cell lysis. In addition, preserving the nanocellulose gel colloidal stability during blood typing is critical to ensure a homogeneous matrix, guaranteeing consistent cell separation. Translucid, nanocellulose hydrogels allow the accurate determination of agglutinates along the medium and show the occurrence of cell lysis. Additives are required to prevent haemolysis while preserving gel stability. A series of model additives varying in chemical composition and concentration was investigated to optimize blood typing and better understand the driving mechanisms. A monosaccharide (glucose) and disaccharide (trehalose), salts (NaCl and NaOAc), an amino acid (glycine) and a neutral oligomer (PEG) were studied. We achieved reliable blood typing tests presenting well-defined results, clearly differentiating RBCs agglutination from non-agglutination, to visually report strong positive and negative test, respectively. Glycine–nanocellulose gel performed the best for forward typing. This amino acid regulates haemolysis through balancing osmotic pressure to human blood plasma levels and increases cell viability without influencing gel colloidal stability. RBCs acquired biconcave and echinocytic morphologies which are beneficial to their proper centrifugation along the gel column. Salts, such as NaCl and NaOAc, cannot be used with the standard gel as they disrupted electrostatic stabilisation leading to gel collapse. However, once nanocellulose is chemically crosslinked, saline solutions can properly balance tonicity without affecting the fibre network. Indeed, crosslinked gels exhibit a stiffer fibre network (yield point 8 times higher), providing the best conditions for reliable and reproducible reverse blood typing. RBC acquired spherical and biconcave shapes in nanocellulose gels with very low levels of haemolysis. To commercialize nanocellulose for blood typing, economic optimization (amount of antibody needed), stability and shelf-life tests (for antibody and gel function) and performance with red blood cells of weak or partial antibody expression are further required. Abnormal red blood cells, such as sickle or microcytic, must be analysed accordingly, as well as each existing blood group system evaluated independently. This study however presents the immense potential of nanocellulose gel as a novel, low-cost, easy to process, biodegradable and renewable matrix for blood typing applications in particular, and biodiagnostics in general. It represents the first step for a future fully sustainable tests that also includes a renewable gel card replacing the polypropylene currently used. It also provided the basis for the functionalization of nanocellulose with antibodies for a smart matrix for blood typing. Finally, the principles and methodology developed can further be leveraged to utilise this matrix in other studies of cells and their interactions. Supporting Information The supporting information is available online. Evaluation of effects of cellulose carboxylate content, effects of additives at lower concentration, triplicates for blood typing test, rheological analysis for hydrogels containing additives and the method for image analysis are present.


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Acknowledgments This work was funded by the Australian Research Council (ARC) Bioprocessing Advance Manufacturing Industry Research Transformation Hub IH13100016 and ARC LP160100544 with Haemokinesis. Many thanks to Australian Paper for providing the pulp. Thanks to Dr David Bashforth for discussion; Dr Cheang Ly Be and Professor Susan Nilsson for help with osmolality measuring; Dr Clare Henderson for image analysis expertise.

References (1) Mitra, R.; Mishra, N.; Rath, G. Blood groups systems. Indian J. Anaesth. 2014, 58 (5), 524, DOI: 10.4103/0019-5049.144645. (2) Landsteiner, K. Ueber Agglutinationserscheinungen normalen menschlichen Blutes, Braumüller: 1901. (3) Landsteiner, K. Zur Kenntnis der antifermentativen, lytischen und agglutinierenden Wirkungen des Blutserums und der Lymphe. Centralblatt für Bacteriologie 1900, 27, 357-362. (4) Dean, L.; National Center for Biotechnology, I. Blood groups and red cell antigens, NCBI: Bethesda, Md., 2005. (5) Harmening, D. Modern blood banking and transfusion practices, 4th ed. ed.; Philadelphia : F.A. Davis: Philadelphia, 1999. (6) Mujahid, A.; Dickert, F. L. Blood Group Typing: From Classical Strategies to the Application of Synthetic Antibodies Generated by Molecular Imprinting. Sensors (Basel). 2016, 16 (1), 51, DOI: 10.3390/s16010051. (7) Al-Tamimi, M.; Shen, W.; Zeineddine, R.; Tran, H.; Garnier, G. Validation of Paper-Based Assay for Rapid Blood Typing. Anal. Chem. 2012, 84 (3), 1661-1668, DOI: 10.1021/ac202948t. (8) Khan, M. S.; Thouas, G.; Shen, W.; Whyte, G.; Garnier, G. Paper Diagnostic for Instantaneous Blood Typing. Anal. Chem. 2010, 82 (10), 4158-4164, DOI: 10.1021/ac100341n. (9) Su, J.; Al-Tamimi, M.; Garnier, G. Engineering paper as a substrate for blood typing bio-diagnostics. Cellulose 2012, 19 (5), 1749-1758, DOI: 10.1007/s10570-012-9748-7. (10) Lapierre, Y.; Rigal, D.; Adam, J.; Josef, D.; Meyer, F.; Greber, S.; Drot, C. The gel test: a new way to detect red cell antigen-antibody reactions. Transfusion 1990, 30 (2), 109-113, DOI: 10.1046/j.15372995.1990.30290162894.x. (11) Duguid, J. K. Uses of Column Technology in Blood Transfusion. Hematology (Abingdon, U. K.) 1997, 2 (6), 485, DOI: 10.1080/10245332.1997.11746370. (12) Ambuehl, L.; South, S. A sensitive, specific and fast method for unexpected antibody detection using column agglutination technology. Transfusion 1992, 32 (8), S16-S16. (13) Davies, D.; Reis, K.; Chachowski, R.; Jakway, J.; Cupido, A.; Bump, E.; Setcavage, T. Efficacy of no wash antiglobulin (AHG) tests using bead column agglutination technology. Transfusion 1992, 32 (8), S17S17. (14) Langston, M. M.; Procter, J. L.; Cipolone, K. M.; Stroncek, D. F. Evaluation of the gel system for ABO grouping and D typing. Transfusion 1999, 39 (3), 300-305, DOI: 10.1046/j.1537-2995.1999.39399219288.x. (15) Figueiredo, M.; Lima, M.; Morais, S.; Porto, G.; Justiça, B. The gel test: some problems and solutions. Transfusion Med. 1992, 2 (2), 115-118, DOI: 10.1111/j.1365-3148.1992.tb00144.x. (16) Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Cellulose Nanofibers Prepared by TEMPO-Mediated Oxidation of Native Cellulose. Biomacromolecules 2007, 8 (8), 2485-2491, DOI: 10.1021/bm0703970. (17) Nordenström, M.; Fall, A. B.; Nyström, G.; Wågberg, L. The formation of colloidal nanocellulose glasses and gels. Langmuir 2017, 33 (38), 9772–9780, DOI: 10.1021/acs.langmuir.7b01832. (18) Mendoza, L.; Batchelor, W.; Tabor, R. F.; Garnier, G. Gelation mechanism of cellulose nanofibre gels: A colloids and interfacial perspective. J. Colloid Interface Sci. 2018, 509, 39-46, DOI: https://doi.org/10.1016/j.jcis.2017.08.101.



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(19) da Silva Perez, D.; Montanari, S.; Vignon, M. R. TEMPO-Mediated Oxidation of Cellulose III. Biomacromolecules 2003, 4 (5), 1417-1425, DOI: 10.1021/bm034144s. (20) Flory, P. J.; Rehner, J. Statistical Mechanics of Cross‐Linked Polymer Networks II. Swelling. J. Chem. Phys. 1943, 11 (11), 521-526, DOI: 10.1063/1.1723792. (21) Kameneva, V. M.; Repko, M. B.; Krasik, F. E.; Perricelli, C. B.; Borovetz, S. H. Polyethylene Glycol Additives Reduce Hemolysis in Red Blood Cell Suspensions Exposed to Mechanical Stress. ASAIO J. 2003, 49 (5), 537-542, DOI: 10.1097/01.MAT.0000084176.30221.CF. (22) Lang, F.; Busch, G. L.; Ritter, M.; VÖLkl, H.; Waldegger, S.; Gulbins, E.; HÄUssinger, D. Functional Significance of Cell Volume Regulatory Mechanisms. Physiol. Rev. 1998, 78 (1), 247. (23) Khan, S. H.; Ahmad, N.; Ahmad, F.; Kumar, R. Naturally occurring organic osmolytes: From cell physiology to disease prevention. IUBMB Life 2010, 62 (12), 891-895, DOI: 10.1002/iub.406. (24) MacKay, E. M. The Distribution of Glucose in Human Blood. J. Biol. Chem. 1932, 97, 685-689. (25) Jain, S. K. Hyperglycemia can cause membrane lipid peroxidation and osmotic fragility in human red blood cells. J. Biol. Chem. 1989, 264, 21340-21345. (26) Shirey, R. S.; Boyd, J. S.; Ness, P. M. Polyethylene glycol versus low‐ionic‐strength solution in pretransfusion testing: a blinded comparison study. Transfusion 1994, 34 (5), 368-370, DOI: 10.1046/j.15372995.1994.34594249044.x. (27) Lublin, D. M. Universal RBCs. Transfusion 2000, 40 (11), 1285, DOI: 10.1046/j.15372995.2000.40111285.x. (28) Hashemi-Najafabadi, S.; Vasheghani-Farahani, E.; Rasaee, M. A Method To Optimize PEG-Coating of Red Blood Cells. Bioconjug. Chem. 2006, 17 (5), 1288, DOI: 10.1021/bc060057w. (29) Dasgupta, A.; Wahed, A. Chapter 5 - Water, Homeostasis, Electrolytes, and Acid–Base Balance. In Clinical Chemistry, Immunology and Laboratory Quality Control; Elsevier: San Diego, 2014; pp 67-84. (30) Wittels, K. A.; Hubert, E. M.; Musch, M. W.; Goldstein, L. Osmolyte channel regulation by ionic strength in skate RBC. Am J. Physiol. Regul. Integr. Comp. Physiol. 2000, 279 (1), R69. (31) Chamberlin, M. E.; Strange, K. Anisosmotic cell volume regulation: a comparative view. Am. J. Physiol. 1989, 257 (2), C159. (32) Löw, B.; Messeter, L. Antiglobulin test in low-ionic strength salt solution for rapid antibody screening and cross-matching. Vox sang. 1974, 26 (1), 53. (33) Jackson, J. K.; Derleth, D. P. Effects of various arterial infusion solutions on red blood cells in the newborn. Arch. Dis. Child. Fetal Neonatal Ed. 2000, 83 (2), F130. (34) Pakulova, O. K.; Gorobchenko, O. A.; Nikolov, O. T.; Adelyanov, A. V.; Pastukhova, S. Y.; Bondarenko, V. A. The influence of Hofmeister's effect on the osmotic behavior of erythrocytes and on the state of water in their suspension. Materialwiss. Werkstofftech. 2013, 44 (2-3), 167-170, DOI: 10.1002/mawe.201300111.


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Nanocellulose Hydrogel is demonstrated as a low cost, sustainable and highly sensitive medium for gelbased blood typing diagnostics. 224x106mm (96 x 96 DPI)



Figure 1. (a) The gel method for forward blood typing relies on the controlled centrifugation of RBCs and antibodies through a gel column. Positive antigen-antibody interactions promote haemagglutination, where agglutinates become trapped in the upper region of the gel matrix after centrifugation. Conversely, individual RBCs diffuse to the bottom of the tube upon centrifugation, indicating a negative result. (b) 0.1, 0.2 and 0.3 wt.% nanocellulose gels were tested to determine the ideal solids content for reliable blood typing. Gels at 0.1 and 0.2 wt. % did not provide the required fibre network density to distinguish a positive from a negative reaction. 0.3 wt.% cellulose fibres offered the desired separation. However, significant haemolysis was identified. Results represent typical experiments performed in triplicate (n=3) - refer to Figures S1-3. (c) and (d) Optical microscope analysis of agglutinated and individual red blood cells harvested in 0.3 wt.% nanocellulose gel demonstrated the occurrence of antigen-antibody reaction for positives and its absence in negatives. (e) Illustrative sample of 0.3 wt.% nanocellulose hydrogel in a petri dish.


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Figure 2. RBC agglutination for positive and negative tests in 0.3 wt.% nanocellulose gel containing (a-b) non-additives, (c-d) D-glucose, (e-f) trehalose, (g-h) PEG, and (i-j) glycine at 0.45 mmol/g, and 10% (v/v) for PEG. Agglutinated RBCs are trapped in the upper region of the gel column. Individual cells from negative reactions form a pellet at the bottom. Some gel formulations prevent RBC sedimentation. Graph curves indicate the location of red blood cells in the different regions of the gel column, based on analysis of red intensity. Results represent typical experiment performed in triplicate (n= 3) - refer to Figures S4 and S6.



Figure 3. Viscosity curves of nanocellulose gels as a function of nanocellulose concentration. Viscosity–shear rate curves of nanocellulose gels at: (a) different fibre concentration, and upon the addition of (b) glycine at 0.15mmol and 0.45mmol/g against an unmodified gel and a gel diluted with water. (c) Effect of additives on the nanocellulose gels storage modulus (G’) measured from the linear viscoelastic region (LVR) of dynamic strain measurements (25°C, 1Hz). Storage (G’) and loss (G”) modulus versus strain (25°C, 1 Hz) for: (d) 0.3 wt.% nanocellulose gel, and (e) 0.15 and (f) 0.45 mmol/g glycine-containing gels. Measurements were performed at 25°C (n=3).


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Figure 4. (a) Optical microscope analysis of agglutinated and individual red blood cells harvested in nanocellulose gel containing D-glucose, trehalose and glycine at 0.15 and 0.45 mmol/g, and PEG at 5 and 10% (v/v). Results represent experiments performed in triplicate (n= 3). (b) Diameter of individual red blood cells harvested from nanocellulose gels containing D-glucose, trehalose and glycine at 0.15 and 0.45 mmol/g, and PEG at 5 and 10% (v/v). Results represent the mean of experiments performed in sextuplate (n= 6). (c) Flow cytometry analysis of lysed RBC harvested in 0.3 wt.% nanocellulose gel combined to different additives. (d) Osmolality of nanocellulose gels containing D-glucose, trehalose and glycine at 0.15 and 0.45 mmol/g, and PEG at 5 and 10% (v/v). Results represent the mean of experiments performed in triplicate (n= 3, SD = ± 4 mOsmol/kg).



Figure 5. (a) Nanocellulose gels were tested to determine the optimal solids content for reliable reverse blood typing. Gels at 0.1 and 0.2 wt. % did not provide the required fibre network density to distinguish a positive from a negative reaction. A minimum of 0.3 wt.% cellulose fibres offered the desired condition; however, instances of false positives and negatives were noticed. Graphs indicate the intensity analysis of red blood cells in the gel column. (b) Cellulose nanofibers in 0.3 wt.% gels were chemically crosslinked with HDMA, enhancing reproducibility. NaCl 0.9% was added to the gel to balance tonicity. Results represent typical experiments performed in triplicate (n= 3) - refer to Figures S10-13. (c) Optical Microscope analysis of agglutinated and individual red blood cells harvested in 0.3 wt.% standard and crosslinked nanocellulose gel demonstrated the occurrence of antigen-antibody reaction for positives and its absence in negatives. (d) Flow cytometry analysis of lysed RBC harvested in 0.3 wt.% standard and crosslinked nanocellulose gel, in absence and combined to NaCl 0.9%. (e) Osmolality of 0.3 wt.% standard and crosslinked nanocellulose gels containing NaCl. Results represent the mean of experiments performed in triplicate (n= 3, SD = ± 4 mOsmol/kg). (f) Viscosity-Shear rate curves of 0.3 wt.% standard and crosslinked nanocellulose gels (n =3). (g) Stress–shear strain curve for non-crosslinked and crosslinked nanocellulose gels (n =3).


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Nanocellulose Hydrogel for Blood Typing Tests

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